Re: [Histonet] Reason for lengthy primary antibody incubation?

From:

pmarcum@vet.upenn.edu

Hi Patrick,
I agree with Patsy and would add we often extend a very expensive anitbody to
overnight. They do to allow very extended dilutions out to the 1:40,000 is an
extreme example and allows more slides to reacted. Research generally has more
time to do these longer times and the mind set. We are in the position in
research of needing to have a patient's slides completed on the same time day.
I have some antibodies we use on the short clinical type time frame and some we
extend to allow the use of less anitbody in the dilutions. I do use some of
the secondary kits however as we do a large range of animals and no mice or
rats we have to be very careful which animals are used in these kits to avoid
heavy background. Best instance is using the Vector kit when we are doing
equine material as it is a horse serum block and some cross reactivity of the
second antibody. Research is a very different mindset from clinical and
although we have time pressures at times just like clinical. However, we also
the time to workout protocols and make use of some methods not used in clinical
due the time issues.
Pamela Marcun
UPENN New Bolton Center
quoting Patrick Laurie :
> Hello histonet,
>
>
>
> I've been a clinical histotech for about 6 years, and I made a
> transition into research histology about 2 years ago. I hadn't done any
> IHC at my research postion until recently, and I have noticed a couple
> of strange differences. First, there are a wide variety of IHC
> protocols different methods, etc. and I understand the reason for that.
> But some of them have dramatic differences. One collaborating lab has
> an incubation of the primary antibody (Ubiquitin) overnight (16-20 hrs)
> at 4 degrees in a "Hydrated chamber". The primary is quite dilute
> (1:40,000), and another lab adds a similar step for the secondary
> antibody. In my clinical experience, I had primarily used a kit, either
> the Dako Envision or LSAB kit, or the Vector elite kit. My PI, who has
> no IHC experience until now, had me try the protocol with an overnight
> incubation, and now since it worked, he wants me to do every antibody
> from now on like that. He was told by various post docs, etc who have
> been doing research histology that doing a diluted overnight antibody
> will make the primary stick better. I know that research has a lot of
> un-optimized antibodies that might be created in-house or might be for
> another purpose, so a lengthy primary might make sense. But is there
> any reason to do this method for clinically tried and true antibodies?
> I would appreciate all opinions.
>
>
>
> Thanks,
>
>
>
> Patrick Laurie, HT (ASCP)
> Neurogenomics Laboratory
> Benaroya Research Institute
> 1201 9th Ave
> Seattle, Wa 98101
> (206) 341-0681
>
>
>
>
>
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